The goal of the work described in this proposal is to characterize the physiologic role of the type 2 deiodinase (D2) in human skeletal muscle. D2 converts the prohormone thyroxine (T4) to 3,5,3'-triiodothyronine (T3), the active hormone. Our hypothesis is that, in human skeletal muscle, D2 regulates T3- responsive metabolic pathways by modulating the saturation of nuclear T3-receptors (TRs). This hypothesis is founded on data from brain, pituitary, and brown adipose tissue showing that D2-catalyzed T4 to T3 conversion is an important source of intranuclear T3, and on the literature establishing T3 as an important regulator of metabolic processes in skeletal muscle. Our approach to test this hypothesis primarily utilizes commercially available primary human skeletal myocytes in culture (HSMM cells), but also includes a new transgenic "SkD2" mouse with conditional, skeletal muscle-specific expression of D2. In Specific Aim I, we will characterize the regulation of D2 in human skeletal myocytes, and then test the hypothesis that D2- catalyzed T4 to T3 conversion can increase nuclear T3 concentration in these cells enough to alter the expression of T3-responsive genes. In Specific Aim II, we will evaluate the potential for T4, via D2, to modulate intermediary metabolism in these cells. In Specific Aim III, we will create a transgenic mouse with conditional expression of D2 in its skeletal muscle via the tet-off system. We will use this mouse to study the physiologic role of skeletal muscle D2 under normal conditions and during periods of metabolic stress. In addition, we will determine the effect of D2 induction on in vivo glucose homeostasis in collaboration with Dr. Jason Kim of the Yale Mouse Metabolic Phenotypic Center. These studies will provide the candidate with experience in a wide range of molecular biological techniques. He will become an expert in thyroid hormone action, while also gaining valuable exposure in the areas of energetics and metabolism, positioning him well to continue his development towards becoming an independent investigator in endocrinology.